title: Modulating roles of amiloride in irradiation-induced antiproliferative effects in glioblastoma multiforme cells involving Akt phosphorylation and the alternative splicing of apoptotic genes.
abstract: Apoptosis is a key mechanism for enhanced cellular radiosensitivity in radiation therapy. Studies suggest that Akt signaling may play a role in apoptosis and radioresistance. This study evaluates the possible modulating role of amiloride, an antihypertensive agent with a modulating effect to alternative splicing for regulating apoptosis, in the antiproliferative effects induced by ionizing radiation (IR) in glioblastoma multiforme (GBM) 8401 cells. Analysis of cell viability showed that amiloride treatment significantly inhibited cell proliferation in irradiated GBM8401 cells (p<0.05) in a time-dependent manner, especially in cells treated with amiloride with IR post-treatment. In comparison with GBM8401 cells treated with amiloride alone, with GBM8401 cells treated with IR alone, and with human embryonic lung fibroblast control cells (HEL 299), GBM8401 cells treated with IR combined with amiloride showed increased overexpression of phosphorylated Akt, regardless of whether IR treatment was performed before or after amiloride administration. The alternative splicing pattern of apoptotic protease-activating factor-1 (APAF1) in cells treated with amiloride alone, IR alone, and combined amiloride-IR treatments showed more consistent cell proliferation compared to that in other apoptosis-related genes such as baculoviral IAP repeat containing 5 (BIRC5), Bcl-X, and homeodomain interacting protein kinase-3 (HIPK3). In GBM8401 cells treated with amiloride with IR post-treatment, the ratio of prosurvival (-XL,-LC) to proapoptotic (-LN,-S) splice variants of APAF1 was lower than that seen in cells treated with amiloride with IR pretreatment, suggesting that proapoptotic splice variants of APAF1 (APAF1-LN,-S) were higher in the glioblastoma cells treated with amiloride with IR post-treatment, as compared to glioblastoma cells and fibroblast control cells that had received other treatments. Together, these results suggest that amiloride modulates cell radiosensitivity involving the Akt phosphorylation and the alternative splicing of APAF1, especially for the cells treated with amiloride with IR post-treatment. Therefore, amiloride may improve the effectiveness of radiation therapy for GBMs.
authors.affiliation: Department of Radiation Oncology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Jen-Yang
authors.initials: JY
authors.lastname: Tang
authors.forename: Hsueh-Wei
authors.initials: HW
authors.lastname: Chang
authors.forename: Jan-Gowth
authors.initials: JG
authors.lastname: Chang
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D000584:Amiloride / Q000494:pharmacology*; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects* / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004354:Drug Screening Assays, Antitumor; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D005909:Glioblastoma / Q000473:pathology; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011836:Radiation Tolerance / Q000187:drug effects; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D000077022:Survivin; D013997:Time Factors
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D011838:Radiation-Sensitizing Agents; D000077022:Survivin; D000584:Amiloride; C109102:HIPK3 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt